This invention relates to methods of detecting transcriptional activity in a cell using two or more reporter genes.
Reporter gene assays are useful in the study of gene regulatory elements because reporter gene activity, i.e., production of the reporter protein, is directly proportional to transcriptional activity of the regulatory elements of the gene. A reporter gene construct for use in these assays contains one or more gene regulatory elements which are of interest, the minimal sequence requirements for transcription of a functional mRNA and the coding sequence for a reporter protein. Alam, J., et al, Anal. Bioch., 188: 245-254 (1990). Analysis of constructs containing various deletions within the regulatory region enables mapping of regulatory sequences necessary for transcription and cell specific expression.
The reporter protein typically has a unique enzymatic activity or structure which enables it to be distinguished from other proteins present. The activity of the transcribed reporter protein, or quantification of the expressed protein, provides an indirect measurement of gene expression. Reporter gene assays enable the identification of sequences and factors that control gene expression at the transcriptional level. Bronstein, I., et al, BioTechniques, Vol. 17, No. 1, p. 172 (1994). Other uses for reporter gene assays include: identification of sequences and factors that control genes at the translational level, study of mechanisms and factors that influence and alter gene expression levels and drug screening in cell-based assays.
In single reporter gene assays with poor sensitivity it is difficult to distinguish negative results caused by the lack of expression or low level assay sensitivity. This problem can be overcome with assays of greater sensitivity. Multiple gene assays are commonly used to provide controls for efficiency of transfection. In such assays, cells are transfected with a mixture of two separate plasmids, each having a different reporter gene. The expression of one reporter gene is controlled by different regulatory regions being studied while the other reporter gene, acting as a control, is generally constitutively expressed by a standard promoter or enhancer. The activity of the experimental reporter enzyme is normalized to the activity of the control reporter enzyme.
In the known examples of assaying multiple reporter gene expression, a separate aliquot of the sample must be used in a separate assay to test for the activity of each enzyme. Alam, J., et al, Anal. Bioch., 188: 245-254 (1990). The necessity of testing a separate portion for each enzyme decreases the precision of the assay and may introduce experimental errors into the results. Therefore, a multiple reporter gene assay which is sequentially performed on the same aliquot of cell extract would simplify the assay procedure and minimize experimental errors. The use of multiple reporter genes can improve the efficiency of high throughput screening for drug discovery.
To provide relevant experimental information, reporter assays must be sensitive, thus enabling the detection of low levels of reporter protein in cell lines that transfect poorly. The sensitivity of a reporter gene assay is a function of the detection method as well as reporter mRNA and protein turnover, and endogenous (background) levels of the reporter activity.
Commonly used detection techniques use isotopic, calorimetric, fluorometric or luminescent enzyme substrates and immunoassay-based procedures with isotopic or color endpoints. Many of these systems, however, have disadvantages that limit their usefulness in these assays. For example, isotopic substrates and immunoassays are limited by the cost, sensitivity and inconvenience of using radioisotopes. Fluorometric systems require external light sources which must be filtered to discriminate fluorescent signal, thereby limiting the sensitivity and increasing complexity of the detection system. Furthermore, fluorescence from endogenous source can interfere with fluorometric measurements. Colorimetric systems lack the sensitivity desired for sensitive reporter gene assays. Chemiluminescent and bioluminescent assays, on the other hand, have been found to be more rapid and sensitive than calorimetric assays and fluorometric assays. Jain, V. K. and Magrath, I. T., Anal. Biochem., 199: 119-124 (1991). It would be, therefore, desirable to have a multiple reporter gene assay as aforesaid, which uses a luminescent detection system.
A number of genes are currently used as reporter genes including chloramphenicol acetyltransferase (CAT), secreted alkaline phosphatase, luciferase, xcex2-galactosidase, xcex2-glucuronidase, and human growth hormone, among others. Bronstein, I., et al, Anal. Biochem., 219: 169-181 (1994). xcex2-galactosidase and CAT are two of the most widely used reporter genes. See Alam, et al., 1990). xcex2-galactosidase detection is commonly performed with calorimetric substrates which lack sensitivity. Fluorescent substrates are also used to detect xcex2-galactosidase, however, those assays also lacks sensitivity and are limited by background autofluorescence and signal quenching. The most widely used assay for CAT is radioisotopic, exhibits only moderate sensitivity, and suffers from a narrow dynamic range. xcex2-Glucuronidase (GUS) is a very widely used reporter gene in plant genetic research and to a lesser extent in mammalian cells. A common assay for GUS uses a fluorescent substrate, but is limited by background autofluorescence and signal quenching. Luciferase has become a more widely used reporter gene as it is quantitated using a very sensitive bioluminescent assay utilizing the substrate, luciferin.
Sensitive chemiluminescent assays, not limited to reporter gene assays, have been described using dioxetane substrates. Bronstein, U.S. Pat. No. 4,978,614, incorporated herein by reference. These dioxetane substrates emit visible light following enzyme induced degradation. Enhancement of the chemiluminescent degradation of 1,2-dioxetanes by enhancer substances comprised of certain water soluble substances, such as globular proteins that have hydrophobic regions, has been described. Voyta et al., U.S. Pat. No. 5,145,772, incorporated herein by reference. These dioxetane substrates are also used in reporter gene assays for alkaline phosphatase, xcex2-galactosidase, and xcex2-glucuronidase for example. See e.g., Bronstein, I., et al, Anal. Biochem., 219: 169-181 (1994) and citations therein. However, no reporter gene assay using dioxetane substrates has been described in which the products of multiple reporter genes are sequentially quantitated in the same aliquot of cell extract.
Simple, rapid and highly sensitive, combined multiple reporter gene assays to detect commonly used reporter genes which do not use radioisotopes or require external light sources are highly desirable.
It is desirable to have a multiple reporter gene assay in which the reagents enhance the light signal produced by the reporter enzymes. It is also important that the signal from one reporter enzyme in a multiple reporter gene assay does not significantly interfere with the signal from the other reporter enzymes during measurement of their maximum light signal. It would be useful to have an assay which produces enhanced levels of light and therefore increases assay dynamic range and sensitivity and enables the use of a wide variety of instruments.
The method of the present invention provides a rapid, highly sensitive, non-isotopic method for sequentially detecting multiple reporter gene products in a single aliquot of cell extract. The method of the present invention comprises quantifying the activity of a first reporter enzyme by measuring the light signal produced by degradation of a substrate by the first reporter enzyme, and quantifying the activity of a second reporter enzyme by measuring the light signal produced by degradation of a second substrate by the second reporter enzyme, wherein both quantifications are sequentially performed on the same aliquot of sample extract. In one preferred embodiment, the presence of the first substrate enhances the light signal produced by degradation of a second substrate.
In preferred embodiments, the method of the present invention further comprises decreasing the light signal produced from the first reporter enzyme prior to quantifying the activity of the second reporter enzyme. Decreasing the signal from the first reporter enzyme preferably comprises substantially inactivating the first reporter enzyme or decreasing the amount of the first substrate.
In the methods of the present invention, substantially inactivating the first reporter enzyme comprises altering the pH of the aliquot, heating the aliquot to degrade the first enzyme or adding specific reagents to decrease activity of the first enzyme, such as alcohols such as isopropanol or ethanol, surfactants such as cetyl trimethyl ammonium bromide (CTAB) or substrate analogs.
In the methods of the present invention, decreasing the amount of the first substrate comprises adding an additional amount of the first enzyme sufficient to degrade the first substrate.
The reporter enzymes useful in the practice of this invention include luciferase, xcex2-galactosidase, xcex2-glucuronidase, alkaline phosphatase, and secreted placental alkaline phosphatase. Preferably, at least one of the first or second reporter enzymes is a hydrolytic enzyme capable of reacting with a dioxetane substrate. Useful hydrolytic enzymes include alkaline and acid phosphatases, esterases, decarboxylases, phospholipase D, xcex2-xylosidase, xcex2-D-fucosidase, thioglucosidase, xcex2-D-galactosidase, xcex1-D-galactosidase, xcex1-D-glucosidase, xcex2-D-glucosidase, xcex1-D-mannosidase, xcex2-D-mannosidase, xcex2-D-fructofuranosidase, xcex2-D-glucosiduronase, and trypsin. In one preferred embodiment, the first reporter enzyme is luciferase. In preferred embodiments, the second reporter enzyme comprises xcex2-galactosidase. In one particularly preferred embodiment the first reporter enzyme is luciferase and the second reporter enzyme is xcex2-galactosidase.
Preferably, at least one of the first or second substrates is a dioxetane. Dioxetane substrates useful in the present invention include:3-(2xe2x80x2-spiroadamantane)-4-methoxy-4-(3xe2x80x3-phosphoryloxy)phenyl-1,2-dioxetane, disodium salt (AMPPD), or disodium 3-(4-methoxyspiro[1,2-dioxetane-3,2xe2x80x2(5xe2x80x2-chloro)-tricyclo-[3.3.1.13,7]decan]-4-yl]phenyl phosphate (CSPD), 3-(2xe2x80x2-spiroadamantane)-4-methoxy-4-(3xe2x80x3-xcex2-D-galactopyranosyl)phenyl-1,2-dioxetane (AMPGD); 3-(4-methoxyspiro[1,2-dioxetane-3,2xe2x80x2-(5xe2x80x2-chloro)tricyclo[3.3.1.13,7]decan]-4-yl-phenyl-xcex2-D-galactopyranoside (Galacton(trademark)), and 5-chloro-3-(methoxyspiro[1,2-dioxetane-3,2xe2x80x2-(5xe2x80x2-chloro)tricyclo[3.3.13,7]decan-4-yl-phenyl-xcex2-D-galactopyranoside (Galacton-Plus).
In certain embodiments, the method further comprises adding a water soluble polymeric enhancer molecule to enhance the light signal produced by enzymatic degradation of the dioxetane. There can be one enhancer for each dioxetane or the same enhancer can be used for multiple substrates. Polymeric enhancers useful in the practice of the present invention include bovine serum albumin, human serum albumin or polymeric quaternary onium salts. The polymeric quaternary onium salts comprise poly vinylbenzyltrimethylammonium chloride (TMQ), polyvinyl benzyl tributyl ammonium chloride (TBQ), polyvinylbenzyltributylphosphonium chloride, polyvinylbenzyl benzyldimethylammonium chloride (BDMQ) or polyvinyl tributyl sulfonium chloride. Other co-polymers, such as water soluble quarternary ammonium-phosphonium, ammonium-sulfonium and. sulfonium-phosphonium polymers are also useful as enhancer molecules.
In other embodiments, these methods further comprise adding an accelerator solution prior to measuring the second enzyme activity. The accelerator solution is used to substantially inactivate the first reporter enzyme and simultaneously increase the light signal produced by the second enzyme. The accelerator solution comprises a water soluble polymeric enhancer molecule. Preferably the pH of the accelerator is from about 9 to about 12. When the accelerator is added to the aliquot of cell extract it alters the pH of the aliquot. For example, in a preferred embodiment, the accelerator solution is at a pH at about 10.8. When the accelerator is added to the aliquot it increases the pH of the aliquot from about 6.0 to greater than 9.0. The altered pH of the aliquot decreases the activity of the first reporter enzyme and activates the production of signal from accumulated enzymatic product of the degradation of the second substrate. The polymeric enhancers described in the preceding paragraph are used in the accelerator solution.
The method of this invention also provides methods of quantifying the product of more than two reporter genes by measuring multiple enzyme activities in a single aliquot of cell extract. One such method comprises (a) quantifying the activity of a first reporter enzyme in an aliquot of the cell extract by measuring the light signal produced by degradation of a first substrate; (b) quantifying the activity of a second reporter enzyme in the aliquot of the cell extract by measuring the light signal produced by degradation of a second substrate; and (c) quantifying the activity of a third reporter enzyme in the aliquot of the cell extract by measuring the light signal produced by degradation of a third substrate, wherein all quantifications are sequentially performed on the same aliquot of sample extract. In this method, the first, second and third substrates are different and at least one of the substrates is a dioxetane. In certain embodiments, the method of measuring products of more than two reporter genes further comprises decreasing the activity of the reporter enzymes prior to quantifying the activity of the subsequent reporter enzyme. For example, prior to quantifying the activity of the second reporter enzyme, the light signal generated by the first substrate for the first reporter enzyme is decreased. This method further comprises the measurement of the activity of the second reporter enzyme and then further measuring the activity of the third reporter enzyme.
The invention also provides a system for detecting the products of more than one reporter gene in an aliquot of a sample extract, the system comprising: the reagents for quantifying each of two or more reporter enzymes; the substrates for each of the reporter enzymes, wherein at least one of the substrates is a dioxetane; and an accelerator solution containing a water soluble polymeric enhancer molecule. The accelerator solution comprises a water soluble polymeric enhancer molecule at a pH from about 9 to about 12, to induce the signal produced from the second or a third reporter enzyme-substrate reaction and decrease the activity of the first reporter enzyme. The polymeric enhancer comprises bovine serum albumin, human serum albumin or polymeric quaternary ammonium, sulfonium and phosphonium salts. The polymeric quaternary ammonium, sulfonium and phosphonium salts comprise polyvinylbenzyltrimethylammonium chloride (TMQ), polyvinylbenzyltributylammonium chloride (TBQ), polyvinylbenzyl benzyldimethylammonium chloride (BDMQ) polyvinylbenzylsulfonium chlroide or polyvinylbenzyl tributylphosphonium chloride. Other co-polymers, such as ammonium-phosphonium, ammonium-sulfonium and sulfonium-phosphonium quarternary polymers are also useful as enhancer molecules.
One preferred embodiment of the invention provides a method of measuring the products of more than one reporter gene in an aliquot of a sample extract. The method comprises: (a) adding a first substrate which is the substrate of a first reporter enzyme product and a second substrate which is the substrate of a second reporter enzyme product to an aliquot of the cell extract, the first substrate comprising luciferin for detection of the first enzyme luciferase and the second substrate comprising a dioxetane and the second enzyme being a hydrolytic enzyme; (b) measuring the activity of the first reporter enzyme; (c) adding an accelerator solution which substantially inactivates the first reporter enzyme and simultaneously increases the chemiluminescent signal produced from the degraded substrate for the second reporter enzyme by increasing the pH of the aliquot, and (d) measuring the chemiluminescent signal produced from the degraded substrate for the second reporter enzyme in the same aliquot of the cell extract, wherein the presence of the first substrate enhances the light signal produced by degradation of a second substrate. In one preferred embodiment, the first reporter enzyme is luciferase and the second reporter enzyme is xcex2-galactosidase.
The present invention provides a method of detecting the activity of a first and second reporter enzyme sequentially in the same aliquot of cell extract sample rather than using separate aliquots of the cell extract to individually measure each enzyme as has been previously performed in the art. The method of the present invention therefore decreases the likelihood of experimental error, thus enabling more reliable data.